Culture medium and method capable of efficiently amplifying human mesenchymal stem cells and keeping stemness of human mesenchymal stem cells
A technology of bone marrow mesenchyme and culture medium, applied in bone/connective tissue cells, culture process, animal cells, etc., can solve the problems of easy differentiation, aging, loss of multi-directional differentiation potential, etc., and achieve the effect of improving the amplification efficiency
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Embodiment 1
[0046] The effect of single Si on the proliferation of human bone marrow mesenchymal stem cells:
[0047] (1) Prepare CaSiO according to ISO 10993-5 standard 3 Extraction solution. The mass volume ratio of powder and hBMSC special medium is 200mg mL -1 Add the powder to the culture medium, seal it and place it in a constant temperature shaker at 37°C for 24h, centrifuge at 4000rpm for 10min, take the supernatant and filter it through a 0.22μm membrane to obtain 200mg mL -1 CaSiO 3 Extract the stock solution. 200 mgmL -1 CaSiO 3Dilute the extract solution with hBMSC-specific complete medium to 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, 1 / 128, and 1 / 256, and store it in a 4°C refrigerator for later use , and measure the ion concentration (see Table 1) by inductively coupled plasma optical emission spectrometer (ICP-OES);
[0048] (2) Inoculate hBMSCs into 96-well cell culture plates at a density of 800 cells / well, set up 6 parallel well experiments for each concentration, use the c...
Embodiment 2
[0054] Effects of Si-Sr combination and single Sr corresponding to the same concentration on the proliferation of human bone marrow mesenchymal stem cells:
[0055] (1) Prepare CaSiO according to ISO 10993-5 standard 3 , SrSiO 3 and SrO extract. The mass volume ratio of powder and hBMSC special medium is 200mg mL -1 Add the powder to the culture medium, seal it and place it in a constant temperature shaker at 37°C for 24h, centrifuge at 4000rpm for 10min, take the supernatant and filter it through a 0.22μm membrane to obtain 200mg mL -1 CaSiO 3 , SrSiO 3 and SrO leaching stock solution. Measure the ion concentration of the stock solution by inductively coupled plasma optical emission spectrometer (ICP-OES);
[0056] (2) CaSiO 3 The extraction stock solution was diluted with complete hBMSC special medium to Si concentration of 2.59ppm. by CaSiO 3 Leaching stock solution and SrSiO 3 The original solution was mixed, and the Si concentration was diluted to 2.59 ppm with ...
Embodiment 3
[0065] (1) Select the complete medium of the optimal Si (2.59ppm)-Sr (20.20ppm) combined extract as the experimental group, and the complete medium of the single Si (2.59ppm) and single Sr (20.20ppm) extract as the experimental control group, hBMSC complete medium as the blank control group;
[0066] (2) Inoculate the fourth-generation hBMSCs into a 6-well cell culture plate at a density of 2.0*10^5 cells / well, and after 12 hours of cell attachment, replace the medium with 2ml / well containing the optimal Si-Sr combination Concentration and its corresponding concentration of single Si, Sr ion extract complete medium and blank control medium, when the cells reach 80%-90% confluence after 48h, some cells are collected and passaged at a ratio of 1:2 ;
[0067] (3) According to the above method, it has been passed down to the eighth generation, and the cells of the fourth generation, the sixth generation and the eighth generation are collected respectively;
[0068] (4) SYBR Gree...
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